Pathogenicity of Aspergillus Clavatus Produced in a Fungal Biofilm Bioreactor Toward Culex Quinquefasciatus (Diptera: Culicidae)
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J. Pestic. Sci. 39(3), 1–6 (2014) DOI: 10.1584/jpestics.D14-006 Original Article Pathogenicity of Aspergillus clavatus produced in a fungal biofilm bioreactor toward Culex quinquefasciatus (Diptera: Culicidae) Fawrou Seye,1,2,† Thomas Bawin,2,*,† Slimane Boukraa,2 Jean-Yves Zimmer,2 Mady Ndiaye,1 Frank Delvigne3 and Frédéric Francis2 1 Laboratory of Reproductive Biology, Department of Animal Biology, Faculty of Science and Technology, University Cheikh Anta Diop, P.O. Box-5005, Dakar Fann, Senegal 2 Functional and Evolutionary Entomology, Gembloux Agro-Bio Tech, University of Liege, Passage des Déportés 2, P.O. Box-5030 Gembloux, Belgium 3 Bio-Industries/C.W.B.I., Gembloux Agro-Bio Tech, University of Liege, Passage des Déportés 2, P.O. Box B-5030 Gembloux, Belgium (Received January 21, 2014; Accepted May 18, 2014) Many entomopathogenic fungi have been demonstrated to be potential agents for efficiently controlling mosquito populations. In the present study, we investigated a bioreactor system to produce metabolites and conidia by combining technological advan- tages of submerged and solid-state fermentations. The efficiency of fungal products was tested toward mosquitoes. Aspergillus clavatus (Eurotiales: Trichocomaceae) was grown by semi-solid-state fermentation in a bioreactor for up to 7 days. Depending on conidial doses (2.5×107, 5×107, 7.5×107, 10×107 and 12.5×107 conidia/mL), mortality ranged from 37.2±15.0 to 86.3±5.0% toward larvae and from 35.8±2.0 to 85.2±1.5% toward adults. The metabolites (10, 20, 40, 60, 80 and 100% v/v) yielded mortal- ity from 23.7±15.0 to 100.0±0.1% toward larvae, and two sprayed volumes (5 and 10 mL) reached 45.5±1.4 and 75.6±2.6% mortality, respectively, toward adults. © Pesticide Science Society of Japan Keywords: biological control, entomopathogenic fungi, Aspergillus clavatus, biofilm bioreactor, solid-state fermentation, sub- merged fermentation. efficient against mosquitoes.8,9) The possibility of using Asper- Introduction gillus niger metabolites against Cx. quinquefasciatus, Anopheles Mosquitoes (Diptera: Culicidae) are permanent blood sucking stephensi and Aedes aegypti larvae has also been shown.10) vectors of diseases such as dengue, filariasis, and malaria. Many The development of a reliable and efficient fermentation sys- mosquito species and strains are resistant to insecticides com- tem for large-scale production of insecticidal products is still monly used for their control. Biological agents have become needed. Classical submerged fermentation of filamentous mi- increasingly attractive alternative for mosquito control. Among croorganisms in aqueous suspensions may impair the produc- potential microorganisms, entomopathogenic fungi have been tion of conidia and metabolites of biotechnological interest.11–13) used and have provided most interesting results in several pest By contrast, solid-state fermentation is a promising alternative controls. Conidia are classically applied, but toxic metabolites since conidia and metabolites are generally produced in higher have also been produced in liquid medium and used against quantities when these microorganisms are produced on a solid mosquitoes.1,2) Metarhizium anisopliae and Beauveria bassiana substrate.11–16) For example, Aspergillus oryzae has been reported are promising alternatives to conventional insecticides for elimi- to produce a 500-fold higher yield of recombinant chymosin in nating mosquito vectors.3,4) Recently, many Aspergillus species solid-state fermentation than in submerged fermentation.17) In have commonly been used against mosquitoes.5,6) The larvicidal this context, efficient utilization of agro-industrial residues as effect of Aspergillus flavus and Aspergillus parasiticus metabo- carbon sources has been shown for the mass production of ento- lites for controlling Culex quinquefasciatus has been shown.7) mopathogenic fungi.18–21) However, the absence of free water in- Aspergillus clavatus conidia have also been demonstrated to be duces parameter variations (such as pH, temperature, moisture, dissolved oxygen or CO2) that are difficult to control. A comple- mentarity method between solid- and liquid-state fermentations † These authors contributed equally to this work. 22) * To whom correspondence should be addressed. has been attempted in sequential culture. E-mail: [email protected] A fungal biofilm reactor combining the technological advan- Published online ●●● ●●, ●●●● tages of submerged fermentation (i.e., free water facilitating the © Pesticide Science Society of Japan control of culture parameters) with the biological characteris- 2 F. Seye et al. Journal of Pesticide Science tics found in solid-state fermentation has been established in the present study. Insecticidal activity of conidia and metabolites secreted by an entomopathogenic A. clavatus strain in this cul- ture system was shown against Cx. quinquefasciatus larvae and adults. Materials and Methods 1. Fungal strain An A. clavatus (Eurotiales: Trichocomaceae) strain isolated from the locust cricket Oedaleus senegalensis (Orthoptera: Acrididae) at the Laboratory of Reproductive Biology of the University Cheikh Anta Diop (Dakar, Senegal) and shown to be pathogenic against Anopheles gambiae, Ae. aegypti and Cx. quinquefascia- tus larvae9) was used (accession number MUCL 55275, Belgian Coordinated Collections of Microorganisms, Mycothèque de l’Université Catholique de Louvain (Belgium)). This strain was cultivated on potato dextrose agar (PDA) in Petri dishes and stored at 4°C. 2. Inoculum As a preculture, A. clavatus mycelia were produced in Erlenmey- Fig. 1. Schematic diagram of a 20-L bioreactor system with metal pack- er conical flasks (500 mL) containing 300 mL of distilled water, ing. The packed structure (16 cm height×16 cm diameter) consisted of 1% peptone, 1% yeast extract and 2% glucose sterilized at 120°C a stainless steel cylinder composed of several independent rectangular for 20 min. These cultures were incubated after inoculation for plates surrounded by a broad plate. The cylinder was closed at the bot- 48 hr on a rotary shaker at 140 rpm and 30°C to produce fungal tom by a circular plate. Uniform liquid media were ensured by mixing. pellets. The pump drew the liquid containing the fungal pellets provided from a preculture, and injected them into the packing containing wheat bran as a solid substrate. Biomass immobilization was ensured to avoid liquid vis- 3. Metal structured packing for fungal sporulation cosity. To facilitate pellets fixation and fungal sporulation in a biore- actor, a metal packing was molded. Packing is a cylinder com- posed of several stainless steel corrugated sheets independent containing wheat bran. The mycelial pellets were then carried of each other, as previously described.23) For packing, 28 metal out with the liquid medium and fixed to the molded system. The rectangular plates (16×16 to 2×16 cm) with a 2-mm mesh were temperature was maintained at 30°C by circulating temperature- cut for assembly. The plates were superimposed symmetrically controlled water. Air was continuously supplied to the bioreac- in pairs following the decreasing width of the rectangle in order tor at 3 L/min. The pH was allowed to vary freely and was re- to form a cylinder approximately 16 cm in diameter. All were corded continuously. Cultivation was carried out for 7 days. then surrounded by a broad plate to form a block of cylindri- cal packages 16 cm in height and 16 cm in diameter. One of the 5. Conidia recovery bases was covered by a circular metal to prevent solid substrates After incubation, the packing that contained fungal conidia from falling into the liquid medium. and biomass was removed, and the liquid medium was stored at −80°C. The packing was washed with an aqueous solution 4. Bioreactor system containing 0.05% Tween 80 (0.05% Tween 80 solution) to detach The fermentation run was carried out in a 20-L bioreactor with the conidia. The washing solution was then filtered to discard 6-L working volume. Wheat bran (200 g) was introduced as a impurities and centrifuged (4°C, 3,000 g, 5 min). The conidia carbon source in the cylinder between the metal plates of the were finally suspended in a 0.05% Tween 80 solution and the packing, which was suspended in the Biolafitte bioreactor (Fig. dose was adjusted to 109 conidia per mL using a hemocytometer 1). Peptone and yeast extract (60 g each) were previously intro- (Thoma®). duced in the total working volume of the reactor containing 6 L of distilled water and 0.05 g/L chloramphenicol as a bacte- 6. Filtration of metabolites riostatic agent. The preparation was autoclaved at 120°C. The Liquid medium from the bioreactor was centrifuged (4°C, preculture was aseptically injected into the bioreactor using a 7,500 g, 30 min) to eliminate impurities. Medium was then fil- syringe. With a peristaltic pump (120 rpm), the culture medium tered with ultrafiltration membranes (Prep/Scale-TFF-1 Car- was continuously stirred (connections made with silicone tubing tridge (Millipore) 5.8 cm in diameter and 15.2 cm long with with an internal diameter of 5 mm) and injected on the packing polyethersulfone membrane filtration (column 1 ft2 cartridge)) Vol. 39, No. 3, 000–6 (2014) Aspergillus metabolites and conidia produced in a biofilm bioreactor 3 to remove unconsumed nutrients from the culture medium. was sprayed on the adults. In parallel, 5 and 10 mL of concen- Final concentrated metabolites (800 mL) with high molecular trated metabolites were sprayed directly on the groups of 20 weight products (MW>100,000) were obtained and stored at adults previously introduced into separate cages and kept for 3 −80°C. days. For control, distilled water was sprayed on the adults. 8.3 Statistical analysis 7. Mosquito rearing Four replicates were performed for each treatment for different Cx. quinquefasciatus (S-Lab) adults were reared in 50-×50- durations with new independent fermentation products. Mortal- ×50-cm cages and fed with a 10% sucrose solution. Blood meal ity was recorded daily for three days and corrected using Ab- was made using the Hemotek feeding system.